Dithranol Targets Keratinocytes, Their Crosstalk with Neutrophils And

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RESEARCH ARTICLE

Dithranol targets keratinocytes, their crosstalk with neutrophils and inhibits the IL-36 inflammatory loop in psoriasis Theresa Benezeder1†, Clemens Painsi2†, VijayKumar Patra1, Saptaswa Dey1, Martin Holcmann3, Bernhard Lange-Asschenfeldt2, Maria Sibilia3, Peter Wolf1*

1Department of Dermatology, Medical University of Graz, Graz, Austria; 2State Hospital Klagenfurt, Klagenfurt am Wo¨ rthersee, Austria; 3Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria

Abstract Despite the introduction of biologics, topical dithranol (anthralin) has remained one of the most effective anti-psoriatic agents. Serial biopsies from human psoriatic lesions and both the c-Jun/JunB and imiquimod psoriasis mouse model allowed us to study the therapeutic mechanism of this drug. Top differentially expressed genes in the early response to dithranol belonged to keratinocyte and epidermal differentiation pathways and IL-1 family members (i.e. IL36RN) but not elements of the IL-17/IL-23 axis. In human psoriatic response to dithranol, rapid decrease in expression of keratinocyte differentiation regulators (e.g. involucrin, SERPINB7 and SERPINB13), antimicrobial peptides (e.g. ß-defensins like DEFB4A, DEFB4B, DEFB103A, S100 proteins like S100A7, S100A12), chemotactic factors for neutrophils (e.g. CXCL5, CXCL8) and neutrophilic infiltration was followed with much delay by reduction in T cell infiltration. Targeting keratinocytes rather than immune cells may be an alternative approach in particular for topical anti-psoriatic treatment, an area with high need for new drugs. *For correspondence: [email protected] †These authors contributed equally to this work Introduction With the development and market introduction of biologics, much progress has been made in recent Competing interests: The years in the systemic treatment of psoriasis. Currently, targeted therapy with antibodies against IL- authors declare that no 17 or IL-23 exhibits high efficacy and allows complete or almost complete clinical clearance of psori- competing interests exist. asis lesions in a high percentage of cases (Blauvelt et al., 2017; Langley et al., 2014; Papp et al., Funding: See page 24 2017; Reich et al., 2018). However, patients with limited body area involvement (i.e. mild forms of Received: 17 March 2020 psoriasis) who represent the majority of psoriasis patients with up to 90% (Stern et al., 2004) have Accepted: 18 May 2020 been neglected. Not much innovation has occurred in the past few years on topical treatment of Published: 02 June 2020 psoriasis that is mainly prescribed to these patients. Steroids, vitamin D3 and vitamin A analogues are most commonly used as topical agents in such patients, but besides changes in their pharmaceu- Reviewing editor: Ulrich Mrowietz, Christian-Albrechts- tical formulation, they have not been developed further in recent years (de Korte et al., 2008; Universita¨ t zu Kiel, Kiel, Germany Krueger et al., 2001; Langley et al., 2011; Schaarschmidt et al., 2015). Short courses of dithranol (1,8-dihydroxy-9-anthracenone or anthralin) have been successfully used as intermittent topical treat- Copyright Benezeder et al. ment of psoriasis since 1916 (Nast et al., 2012; Sehgal et al., 2014; Vleuten et al., 1996). Despite This article is distributed under its numerous disadvantages like brown staining and irritation of perilesional skin, dithranol has the terms of the Creative Commons Attribution License, remained one of the most effective topical treatment modalities in psoriasis. Analogous to the most which permits unrestricted use recent generation of biologics (including anti-IL-17 and anti-IL-23 antibodies), dithranol delivers and redistribution provided that PASI75 rates in 66–82.5% of patients with fast action and clearance of skin lesion within very few the original author and source are weeks (Keme´ny et al., 1990; Painsi et al., 2015b; Sehgal et al., 2014; van de Kerkhof, 2015). credited. Although dithranol has been used for many years, its exact mechanism of action has remained

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 1 of 31 Research article Immunology and Inflammation largely unknown (Holstein et al., 2017; Keme´ny et al., 1990; Kucharekova et al., 2006; Sehgal et al., 2014). With the aim of unraveling dithranol’s therapeutic mechanisms and to possibly uncover new targets for topical treatment of psoriasis, we conducted a clinical trial and employed several mouse models including the c-Jun/JunB knockout model (Zenz et al., 2005) and the imiquimod psoriasis model (van der Fits et al., 2009) in order to address this issue and elucidate dithranol’s effects. In this study, we demonstrate that dithranol exerts its anti-psoriatic effects by directly targeting keratinocytes and their crosstalk with neutrophils, as well as disrupting the IL-36 inflammatory loop. Con- sistent with this finding, we observed that dithranol’s therapeutic activity was completely independent of its pro-inflammatory effect mainly on perilesional skin, thus overthrowing the long- believed paradigm that dithranol-induced irritation is crucial for its anti-psoriatic action and unraveling irritation merely as a bystander effect of treatment.

Results Topical dithranol leads to fast reduction in PASI score linked to decrease in epidermal hyperproliferation and delayed reduction of inflammatory infiltrate in psoriatic skin As depicted in Figure 1C (and Figure 1—figure supplement 1), dithranol did lead to a fast reduction of psoriatic skin lesions in most of the 15 patients of the study, as determined by psoriasis area and severity index (PASI) and local psoriasis severity index (PSI) of marker lesions. As shown in Table 1, the mean decrease in PASI score was 58% after 2–3 weeks, confirming its high clinical efficacy (Painsi et al., 2015a; Painsi et al., 2015b; Swinkels et al., 2004). Remarkably, dithranol treatment did lead to a visible inflammatory response only at perilesional skin sites, but not within psoriatic plaques and there was no correlation between dithranol-induced erythema and its anti-psoriatic effect (Figure 1—figure supplement 2). The clinical response was confirmed by the results of histological analysis of skin biopsies taken throughout the dithranol treatment course (Figure 1A and B). Dithranol application led to a significant reduction in epidermal hyperplasia (as measured by thickness of epidermis). Intriguingly, there was no significant change in dermal infiltrate score during treatment. At the follow-up visit, hyperplasia of the epidermis was reduced further and cellular infiltrate in the dermis was significantly diminished (Figure 2).

Clinical response to dithranol in psoriasis patients is linked to I) fast upregulation of keratinization genes and downregulation of neutrophil chemotactic genes and neutrophilic infiltration followed by II) delayed downregulation of inflammatory response-related genes and proteins Dithranol slowly diminished CD3, CD4, FoxP3 and CD8 cell counts in the dermis as evidenced by unaffected cell numbers early on during the treatment course and significant reduction only at the follow-up visit (4–6 weeks after end of treatment) (Figure 2). The response of T cells in the epidermis occurred a little earlier, with significant reduction present already at the end of treatment (week 2–3) (Figure 2). In agreement with other studies (Holstein et al., 2017; Swinkels et al., 2002a; Vleuten et al., 1996; Yamamoto and Nishioka, 2003), we found a fast decrease in epidermal hyperproliferation (epidermal thickness, Ki-67 and CK16 staining) at end of treatment (week 2–3). Neutrophil numbers (as assessed by myeloperoxidase staining) in epidermis and dermis were also significantly reduced at that time point. An increase in the number of Langerhans cells in the epidermis (as indicated by CD1a staining) was detected at the follow-up visit. Microarray analysis comparing lesional skin at day 6 of treatment with lesional skin samples at baseline revealed that dithranol led to differential expression of 62 genes including 17 genes with reduced and 45 genes with increased expression. Among these differentially expressed genes (DEGs), there was a significant upregulation of genes involved in keratinization and keratinocyte differentiation (e.g. KRT2, LCE1C, KRT73, LCE1A) and establishment of skin barrier (e.g. FLG2, HRNR, FLG). Furthermore, dithranol downregulated genes of antimicrobial peptides (AMPs) such as ß- defensin-2 (DEFB4A, DEFB4B) and chemoattractants for neutrophils (e.g. CXCL5, CXCL8, PPBP1, TREM1)(Table 2).

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 2 of 31 Research article Immunology and Inflammation

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Figure 1. Dithranol leads to a fast reduction of psoriatic skin lesions as determined by local psoriasis severity index (PSI) of marker lesions. (A,B) 15 psoriasis patients were treated with dithranol. Skin biopsies were taken from marker lesions at multiple timepoints: 1a = lesional skin at baseline, 2 = lesional skin at day 6, 3 = lesional skin at end of treatment, week 2–3, 4 = lesional skin at follow-up, 4–6 weeks after end of treatment. In addition, non-lesional skin at baseline (1b) was sampled. (C) Representative images of lesional psoriatic skin and local psoriasis severity index (PSI; sum of erythema (0–4), induration (0–4) and scaling (0–4); 0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe) at different time points. The online version of this article includes the following source data and figure supplement(s) for figure 1: Figure supplement 1. Psoriasis area and severity (PASI) score at baseline (day 0), early during treatment (day 6), at end of treatment (week 2–3) and at follow-up (4–6 weeks after end of treatment) for all 15 patients (A1–A15) treated with dithranol. Figure supplement 1—source data 1. Values displayed in graph shown in Figure 1—figure supplement 1. Figure supplement 2. Lesional and perilesional erythema does not correlate with reduction in PASI score.

At the end of treatment (week 2–3), dithranol had deregulated a total of 453 genes, with downregulation of 325 DEGs and upregulation of 128 genes. Compared to day 6, expression of various genes involved in keratinocyte differentiation decreased further upon dithranol treatment and other AMPs (e.g. S100A7A, S100A12, DEFB103A) and genes involved in neutrophil-mediated inflammatory responses significantly diminished in their expression. With delay, dithranol also lowered expression of inflammatory response-related genes (e.g. IL1B, IL17, IL22, IL36A, IL36G, IL36RN) only at the end of treatment (week 2–3) (Table 3). Notably, genes involved in T-cell activation were not differentially expressed in the observation period (during dithranol up to week 2–3). To verify a subset of differentially expressed genes from the microarray data, we performed nCounter Nanostring analysis on 80 target and four reference genes. Ratios of microarray target genes strongly correlated with those obtained from Nanostring analysis at the early (day 6) (r = 0.8830) and late time point examined at the end of dithranol treatment (week 2–3) (r = 0.8859) (Supplementary file 2). Comparing day 6 vs. baseline, we found gene ontology (GO) terms related to keratinization (e.g. keratinocyte differentiation, establishment of skin barrier) and neutrophil chemotaxis (e.g. neutrophil migration, regulation of neutrophil migration) among the most significant GO groups (Table 4). Top significantly enriched GO terms at week 2–3 vs. baseline were related to immune response (e.g. inflammatory response, cytokine secretion) and differentiation of keratinocytes (e.g. epidermis development, keratinization) (Table 5). At follow-up (4–6 weeks after end of treatment), 10 of 13 patients

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 3 of 31 Research article Immunology and Inflammation

Table 1. Psoriasis area and severity index (PASI) and psoriasis severity index (PSI) from 15 psoriasis patients.

Parameter Time point Baseline Day 6 End of treatment Follow-up PASI mean ± SD 13.6 ± 10.3 9.0 ± 6.3 5.1 ± 3.8 5.7 ± 6.7 (p<0.0001)* (p=0.0001)* (p=0.0002)* %reduction, mean ± SD (range) - 32.9 ± 8.0 57.5 ± 9.5 56.1 ± 23.3 (16.7–44.3) (41.8–74.2) (3.1–81.0) Local PSI mean ± SD 6.7 ± 1.1 3.3 ± 1.6 2.0 ± 1.5 1.6 ± 1.3 (p<0.0001)* (p<0.0001)* (p<0.0001)* %reduction, mean ± SD (range) - 52.6 ± 21.9 69.0 ± 23.9 76.3 ± 18.3 (14.3–100) (16.7–100) (37.5–100)

*P value was determined using Wilcoxon test comparing indicated value to baseline. The online version of this article includes the following source data for Table 1: Source data 1. Values displayed in Table 1.

showed >50% reduction in epidermal thickness and >75% reduction in Ki67 staining. These 10 patients were classified as histological responders. Microarray gene expression analysis at end of treatment (week 2–3) revealed that 131 genes were differentially expressed in histological responders compared to non-responders, predicting histological outcome for the follow-up time point 4–6 weeks after the end of treatment. Using gene ontology enrichment analysis of these DEGs, we found pathways like keratinocyte differentiation, cornification and keratin filament formation among the top 20 GO terms (Supplementary file 3).

Topical application of dithranol ameliorates psoriasis-like skin lesions in c-Jun/JunB knockout mice by directly targeting keratinocyte genes and inhibiting IL36RN To study the effect of dithranol in a keratinocyte-based psoriatic mouse model, we used c-Jun/JunB knockout mice (treatment protocol shown in Figure 3A). Topical dithranol application strongly reduced psoriatic lesions as measured by macroscopic overall ear thickness and microscopic epidermal thickness in this genetic model of psoriasis based on inducible epidermal deletion of the AP1 transcription factors c-Jun/JunB (Glitzner et al., 2014; Zenz et al., 2005; Figure 3B–D). There was no difference in the outcome to dithranol with regard to different concentrations series, therefore, data was pooled for certain analyses as indicated (Figure 3). Gene expression profiling using Clar- iom S mouse microarray showed that among the top 45 differentially regulated genes, dithranol downregulated genes belonging to the group of late cornified envelope genes (Lce6a, Lce1i, Lce1g, Lce1f), as well as hornerin (Hrnr)(Table 6). Gene ontology enrichment analysis of all differentially expressed genes (fold change >1.5 and p-value<0.05) revealed skin development, epidermis development, epithelial cell differentiation and keratinization among the top 20 significantly enriched pathways (Table 7). Associated genes found were for example Flg2, Ivl, Hrnr, Lor, Krt2, Casp14, Lce1c, Serpinb7 and Serpbinb13, among others. We then compared DEGs from psoriasis patients treated with dithranol to DEGs from dithranol-treated c-Jun/JunB knockout mice (Figure 4). In total, the microarray assay allowed us to compare 300 DEGs in dithranol-treated psoriasis patients (week 2–3 vs. day 0) and 18 DEGs from day 6 vs. day 0 with 336 murine DEGs from dithranol-treated c-Jun/JunB knockout mice. Notably, comparing DEGs from day 6 vs. day 0 with murine DEGs, we found an overlap of 11 genes. Among these genes were CASP14, a non-apoptotic caspase involved in epidermal differentiation and FLG2, HRNR, KRT2, LCE1C and SERPINB12, involved in keratinocyte differentiation and epidermis development (Bergboer et al., 2011; Henry et al., 2011; Henry, 2012; Kuechle et al., 2001; Sandilands et al., 2009; Sivaprasad et al., 2015; Toulza et al., 2007). Even more strikingly, the overlap between DEGs from week 2–3 vs. day 0 and murine DEGs comprised 20

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 4 of 31 Research article Immunology and Inflammation

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Figure 2. Histological and immunohistochemical analysis of lesional skin before (day 0), during (day 6), at end of treatment (week 2–3) and 4–6 weeks after ending treatment (follow-up). (A) Representative H and E images, epidermal thickness and cellular infiltrate scoring. In treated psoriatic lesions, epidermal thickness was significantly decreased at week 2–3 and at follow-up. Infiltrate score (0 = none, 0.5 = none/low, 1 = low, 1.5 = low/moderate, 2 = moderate, 2.5 = moderate/high, 3 = high infiltration of immune cells) was significantly higher in untreated psoriatic lesions compared to non- Figure 2 continued on next page

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 5 of 31 Research article Immunology and Inflammation

Figure 2 continued lesional skin (NL) and healthy controls (HC). In treated psoriatic lesions, infiltrate score was significantly decreased at follow-up. (B) Ki67 staining in epidermis was significantly reduced at week 2–3 and follow-up. (C) CK16 staining in epidermis was significantly reduced at week 2–3 and follow-up. (D– I) Representative IHC images and mean cell counts for epidermis and dermis. Neutrophil cell counts were significantly reduced at week 2–3 and follow- up in epidermis and dermis (D). Langerhans cell (CD1a+) numbers in epidermis were significantly increased at follow-up (E). Epidermal FoxP3 (H) and CD8 (I) cell counts were significantly reduced at week 2–3. Dermal CD3 (F), CD4 (G), and FoxP3 (H) positive cell counts display significant reduction at follow-up. One-way ANOVA with Dunnet’s multiple comparisons test was used for statistics. Bars represent mean ± SD; *p0.05; **p0.01; ***p0.001; ****p0.0001; scale bar = 100 mm. The online version of this article includes the following source data for figure 2: Source data 1. Values displayed in bar plots shown in Figure 2.

genes including IL36RN, IVL, SERPINB7 and SERPINB13, that were all increased in lesional skin at baseline compared to non-lesional skin and downregulated by dithranol treatment. Findings from microarray analysis were verified by RT-PCR (Figure 4—figure supplement 1). The group of genes encoding serpins, as well as involucrin have been shown to play a role in keratinocyte differentiation (Henry, 2012; Toulza et al., 2007) and have been associated with psoriasis (Roberson and Bow- cock, 2010; Sua´rez-Farin˜as et al., 2012; Wolf et al., 2012), belonging to the ‘psoriasis transcriptome’ identified by Tian et al., 2012. In addition, dithranol downregulated expression of elevated IL36A and IL36G in human psoriatic skin and IL36B (Il1f8) in c-Jun/JunB psoriatic skin. To substanti- ate dithranol’s effect on keratinocytes, we employed the mouse-tail test, a traditional model to quantify the effect of topical anti-psoriatics on keratinocyte differentiation by measuring degree of orthokeratosis versus parakeratosis (Bosman et al., 1992; Sebo¨k et al., 2000; Wu et al., 2015). We found a strong increase in percentage of orthokeratosis (from 18.8 to 63.4%) reflecting dithranol’s keratinocyte differentiation-inducing activity (Figure 3—figure supplement 1), consistent with previous work (Bosman et al., 1992; Hofbauer et al., 1988; Sebo¨k et al., 2000; Wrench and Britten, 1975). Next, we performed RT-PCRs of a selected panel of keratinocyte differentiation markers, AMPs and inflammatory markers (based on our microarray data) of dithranol-treated murine tail skin. We found a strong upregulation of keratinization markers (Flg, Krt16, Serpinb3a) and several AMPs (Lcn2, S100a8, S100a9, Defb3))(Figure 3—figure supplement 2). Interestingly, dithranol downregulated expression of the antimicrobial peptide Camp/LL37, as well as Cxcl5, a chemotactic factor for neutrophils. In contrast to the effects of dithranol in the c-Jun/JunB model and mouse-tail test, this agent had no therapeutic capacity in the immunologically mediated imiquimod (IMQ) mouse model, which is often referred to as a psoriatic-like skin inflammation model (van der Fits et al., 2009). Indeed, dithranol treatment worsened psoriatic lesions in that model. Overall skin thickness was significantly enhanced, consistent with an increase in epidermal thickness and worsened inflammation (as measured by cellular infiltrate score) in mice treated with both IMQ and dithranol compared to IMQ- treated mice (Figure 3—figure supplement 3). Different set-ups (i.e. simultaneous treatment with dithranol and IMQ and pre-treatment with IMQ for 5 days followed by dithranol treatment) were tested, but similar effects were observed (data not shown). Taken together, these results demonstrate that dithranol primarily targets keratinocytes and only has delayed effects on other immune cells such as T cells belonging to the IL-17/IL-23 axis in psoriatic skin.

Discussion This study demonstrates that topical dithranol directly targets keratinocytes (in particular their differentiation regulators and AMPs), keratinocyte-neutrophil crosstalk and inhibits the IL-36 inflammatory loop in psoriasis, thus unraveling after over 100 years of use, the therapeutic mechanism of one of the most effective topical treatments of psoriasis. Dithranol significantly diminished mRNA expression of pro-psoriatic IL-1 family members (IL36A, IL36G and IL36RN)(Supplementary file 1 and Fig- ure 4). At day 6 after start of dithranol treatment, PASI had decreased by 33%, but at week 2–3 we saw a reduction of 58%, an effect that was paralleled by reduced expression of IL-36-related genes in psoriasis patients. The therapeutic importance of reduction of IL-1 family members in human skin was substantiated by results generated in the keratinocyte-based c-Jun/JunB mouse psoriasis model (Zenz et al., 2005). Expression of IL36RN (murine Il1f5) and IL36B (Il1f8) was significantly reduced in

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Table 2. Top 45 differentially regulated genes (p-value<0.05, fold change >1.5) in dithranol-treated lesional skin after 6 days compared to baseline from 15 patients with psoriasis. Day 6 vs. baseline Probe set ID Gene symbol Gene title (fold change) 16693318 FLG2 Filaggrin family member 2 2.89 16693303 HRNR Hornerin 2.64 16903552 NEB Nebulin 2.18 16765017 KRT2 Keratin 2 2.04 16693308 FLG Filaggrin 1.97 16761221 CLEC2A C-type lectin domain family 2, member A 1.92 16730782 ELMOD1 ELMO/CED-12 domain containing 1 1.91 16735288 OVCH2 Ovochymase 2 1.83 16990787 SPINK7 Serine peptidase inhibitor, 1.78 Kazal type 7 (putative) 16852824 SERPINB12 Serpin peptidase inhibitor, 1.74 clade B (ovalbumin), member 12 16693341 LCE1C Late cornified envelope 1C 1.71 16693249 THEM5 Thioesterase superfamily member 5 1.70 16921644 MIRLET7C MicroRNA let-7c 1.69 16670681 ANXA9 Annexin A9 1.68 16821186 CLEC3A C-type lectin domain family 3, member A 1.68 16859090 CASP14 Caspase 14 1.68 16765005 KRT73 Keratin 73 1.66 16945497 COL6A5 Collagen, type VI, alpha 5 1.66 16976615 SULT1E1 Sulfotransferase family 1E, 1.65 estrogen-preferring, member 1 16898858 CD207 CD207 molecule, langerin 1.63 16861126 UPK1A Uroplakin 1A 1.60 16704475 FAM35DP Family with sequence similarity 35, 1.60 member A pseudogene 17085015 FRMPD1 FERM and PDZ domain containing 1 1.59 16817034 CHP2 Calcineurin-like EF-hand protein 2 1.56 16671082 LCE1A Late cornified envelope 1A 1.55 16671037 LCE2D Late cornified envelope 2D 1.55 16748835 PIK3C2G Phosphatidylinositol-4-phosphate 1.54 3-kinase, catalytic subunit 17039517 LY6G6C Lymphocyte antigen six 1.53 complex, locus G6C 16673713 FMO2 Flavin containing monooxygenase 2 1.52 (non-functional) 16812344 BCL2A1 BCL2-related protein A1 À1.52 16968213 ANXA3 Annexin A3 À1.54 17104519 RNY4P23 RNA, Ro-associated Y4 pseudogene 23 À1.55 16948835 MIR1224 MicroRNA 1224 À1.55 16815310 TNFRSF12A Tumor necrosis factor À1.60 receptor superfamily, member 12A 17015084 SERPINB1 Serpin peptidase inhibitor, À1.67 clade B (ovalbumin), member 1 17106398 SLC6A14 Solute carrier family 6 À1.69 (amino acid transporter), member 14 16693375 SPRR2F Small proline-rich protein 2F À1.69 Table 2 continued on next page

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 7 of 31 Research article Immunology and Inflammation Table 2 continued

Day 6 vs. baseline Probe set ID Gene symbol Gene title (fold change) 17065458 DEFB4A Defensin, beta 4A À1.75 17074361 DEFB4B Defensin, beta 4B À1.77 16698947 RNU5A-8P RNA, U5A small nuclear 8, pseudogene À1.78 16976827 CXCL5 Chemokine (C-X-C motif) ligand 5 À1.81 16976821 PPBP Pro-platelet basic protein À1.82 (chemokine (C-X-C motif) ligand 7) 17019056 TREM1 Triggering receptor expressed À1.99 on myeloid cells 1 17050251 SLC26A4 Solute carrier family 26 À2.19 (anion exchanger), member 4 16967771 CXCL8 Chemokine (C-X-C motif) ligand 8 À3.36

dithranol-treated psoriatic c-Jun/JunB lesions compared to controls (Figure 4) and among the top differentially expressed genes in our human and mouse dataset were genes involved in keratinocyte and epidermal differentiation (Table 4, Table 7). Dithranol strongly reduced mRNA expression of the keratinocyte differentiation regulator involucrin (IVL) and members of the serpin family (SERP- BINB7, SERBINB13) both in lesional skin of patients and c-Jun/JunB knockout mice (Figure 4). More- over, dithranol downregulated expression of AMPs such as ß-defensins (DEFB4A and DEFB4B) produced by keratinocytes (Liu et al., 2002; Schro¨der and Harder, 1999) within 6 days and chemotactic factors for neutrophils (such as CXCL5 and CXCL8)(Albanesi et al., 2018; Table 2) and neutrophilic infiltration (as determined by MPO staining) within 2–3 weeks of treatment in human psoriatic skin (Figure 2). Surprisingly, there were no significant changes in overall T cell numbers including CD4+ and CD8 + T cells, as well as FoxP3 positivity indicative for regulatory T cells in the skin in the early phase (within 6 days) during dithranol treatment. Reflecting dithranol’s primary effect on the epidermal compartment, the reduction of T cell numbers in the epidermis preceded that in the dermis. Whereas dithranol had decreased T cell counts in the epidermis at week 2–3, an effect on T cell numbers in the dermis was only evident at the follow-up visit, 4–6 weeks after the end of dithranol treatment (Figure 2), in agreement with previous reports favoring dithranol’s effect on keratinocytes (Holstein et al., 2017; Swinkels et al., 2002a; Vleuten et al., 1996; Yamamoto and Nishioka, 2003). Vleuten et al., 1996 and Swinkels et al., 2002a applied immunohistochemistry to analyze differentiation and proliferation markers as well as T cell numbers in the skin and observed, as we did, a decrease in keratin 16, restoration of filaggrin and a decrease of Ki67 in the epidermis after 2 weeks of dithranol treatment. However, their data on the effect of dithranol on dermal T cell numbers at 4 weeks was controversial (Swinkels et al., 2002a; Vleuten et al., 1996). The group of Eberle recently tested the effects of dithranol using primary keratinocytes, a 3D psoriasis tissue model and some biopsy samples from psoriasis patients and reported on a reduction in Ki67 and keratin 16 positive cells in the epidermis using immunostaining and an inhibition of the antimicrobial peptide DEFB4 using qPCR analysis. However, based on their observations, they concluded that dithranol’s anti-psoriatic effects cannot be explained by direct effects on keratinocyte differentiation or cytokine expression (Holstein et al., 2017). Our genome-wide expression analysis indicates that dithranol primarily targets keratinocytes and that this is crucial for response to treatment, considering that differentially regulated genes in histological responders compared to non-responders belonged to pathways like keratinocyte differentiation, cornification and keratin filament formation (Supplementary file 3). The importance of dithranol’s direct effect on keratinocytes has been further substantiated by our findings generated using the mouse-tail model, a simple in vivo model to analyze effects of topical preparations on keratinocyte differentiation and parakeratosis (Bosman et al., 1992; Sebo¨k et al., 2000; Wu et al., 2015). Similar to previous studies (Bosman et al., 1992; Hofbauer et al., 1988; Sebo¨k et al., 2000;

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Table 3. Top 45 differentially regulated genes in dithranol-treated lesional skin at end of treatment compared to baseline from 15 patients with psoriasis (p<0.05, fold change >1.5). Probe set ID Gene symbol Gene title End of treatment vs. baseline (fold change) 16947045 AADAC Arylacetamide deacetylase 3.07 16765005 KRT73 Keratin 73 2.31 16976868 BTC Betacellulin 2.27 16924792 CLDN8 Claudin 8 2.11 17125092 CNTNAP3 Contactin associated protein-like 3 2.09 17097358 SLC46A2 Solute carrier family 46, member 2 2.07 16780133 SLITRK6 SLIT and NTRK-like family, member 6 2.00 16834436 RAMP2 Receptor (G protein-coupled) 1.96 activity modifying protein 2 17123970 ZDHHC11B Zinc finger, DHHC-type containing 11B 1.88 17104313 AR Androgen receptor 1.85 16951140 MIR548I2 MicroRNA 548i-2 1.85 17063366 ATP6V0A4 ATPase, H+ transporting, 1.84 lysosomal V0 subunit a4 16974224 MIR548I2 MicroRNA 548i-2 1.83 16687914 CYP2J2 Cytochrome P450, family 2, 1.82 subfamily J, polypeptide 2 17125106 CNTNAP3B Contactin associated protein-like 3B 1.80 16903552 NEB Nebulin 1.80 17125094 CNTNAP3B Contactin associated protein-like 3B 1.80 16770284 TMEM116 Transmembrane protein 116 1.79 16765041 KRT77 Keratin 77 1.79 17123972 ZDHHC11B Zinc finger, DHHC-type containing 11B 1.78 16688210 MIR3671 MicroRNA 3671 1.78 17125218 CNTNAP3 Contactin associated protein-like 3 1.74 16764923 KRT6A Keratin 6A À3.69 16767261 IL22 Interleukin 22 À3.87 17065453 DEFB103A Defensin, beta 103A À3.94 17074366 DEFB103A Defensin, beta 103A À3.94 16671027 LCE3C Late cornified envelope 3C À4.14 16743751 MMP12 Matrix metallopeptidase 12 À4.42 (macrophage elastase) 16979444 TNIP3 TNFAIP3 interacting protein 3 À4.48 17106398 SLC6A14 Solute carrier family 6 À4.60 (amino acid transporter), member 14 16686734 CYP4Z2P Cytochrome P450, family 4, À4.89 subfamily Z, polypeptide 2 16671144 S100A7A S100 calcium binding protein A7A À5.00 16764907 KRT6C Keratin 6C À5.01 16730157 HEPHL1 Hephaestin-like 1 À5.15 16967831 EPGN Epithelial mitogen À5.31 16842517 NOS2 Nitric oxide synthase 2, inducible À5.38 16693409 S100A12 S100 calcium binding protein A12 À6.12 16813112 RHCG Rh family, C glycoprotein À6.18 16738803 TCN1 Transcobalamin I (vitamin B12 À6.45 binding protein, R binder family) Table 3 continued on next page

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 9 of 31 Research article Immunology and Inflammation Table 3 continued

Probe set ID Gene symbol Gene title End of treatment vs. baseline (fold change) 16924785 CLDN17 Claudin 17 À8.09 16693365 SPRR2C Small proline-rich protein 2C (pseudogene) À8.72 16967771 CXCL8 Chemokine (C-X-C motif) ligand 8 À9.14 17074361 DEFB4B Defensin, beta 4B À9.39 16693375 SPRR2F Small proline-rich protein 2F À10.12 16884602 IL36A Interleukin 36, alpha À10.50

Wrench and Britten, 1975), we observed a strong increase in orthokeratosis after dithranol application in the mouse-tail test, reflecting its keratinocyte differentiation-inducing activity (Figure 3—figure supplement 1). Our transcriptional analysis indicated that the effect of dithranol in the mouse- tail test was linked to a strong upregulation of keratinocyte differentiation markers and several AMPs, while the pro-psoriatic antimicrobial peptide Camp/LL37 was downregulated, as well as Cxcl5, a chemotactic factor for neutrophils (Figure 3—figure supplement 2), but not other pro-psoriatic AMPs (Fritz et al., 2017; Wang et al., 2018) such as Defb3, S100a8 or S100a9. Although the

Table 4. Top 20 significantly enriched pathways as determined by Gene Ontology (GO) enrichment analysis in dithranol-treated lesional skin after 6 days compared to baseline from 15 patients with psoriasis. (GO was done using Cytoscape software [Bindea et al., 2009; Shannon et al., 2003]; a.o. = among others).

GOID GO term P-Value % Associated Genes Associated genes found GO:0001533 Cornified envelope 3.8E-12 10.45 FLG, HRNR, KRT2, LCE1A, LCE1C, LCE2D, SPRR2F GO:0031424 Keratinization 7.7E-12 3.93 CASP14, FLG, HRNR, KRT2, KRT73, LCE1A, LCE1C, LCE2D, SPRR2F GO:0030216 Keratinocyte differentiation 79.0E-12 3.02 CASP14, FLG, HRNR, KRT2, KRT73, LCE1A, LCE1C, LCE2D, SPRR2F GO:0018149 Peptide cross-linking 300.0E-12 9.84 FLG, KRT2, LCE1A, LCE1C, LCE2D, SPRR2F GO:0070268 Cornification 12.0E-9 5.31 CASP14, FLG, KRT2, KRT73, LCE1A, SPRR2F GO:0004168 Receptor CXCR2 binds 1.0E-6 33.33 CXCL5, CXCL8, PPBP ligands CXCL1 to 7 GO:0042379 Chemokine receptor binding 5.4E-6 6.25 CXCL5, CXCL8, DEFB4A, PPBP GO:0045236 CXCR chemokine receptor binding 6.0E-6 18.75 CXCL5, CXCL8, PPBP GO:0061436 Establishment of skin barrier 18.0E-6 13.04 FLG, FLG2, HRNR GO:0033561 Regulation of water loss via skin 21.0E-6 12.00 FLG, FLG2, HRNR GO:0004657 IL-17 signaling pathway 21.0E-6 4.30 CXCL5, CXCL8, DEFB4A, DEFB4B GO:0007874 Keratin filament formation 21.0E-6 4.17 FLG, KRT2, KRT73, SPRR2F GO:0030593 Neutrophil chemotaxis 21.0E-6 4.17 CXCL5, CXCL8, PPBP, TREM1 GO:1990266 Neutrophil migration 27.0E-6 3.85 CXCL5, CXCL8, PPBP, TREM1 GO:0071621 Granulocyte chemotaxis 38.0E-6 3.31 CXCL5, CXCL8, PPBP, TREM1 GO:1902622 Regulation of 45.0E-6 7.50 CXCL5, CXCL8, PPBP neutrophil migration GO:0071622 Regulation of 71.0E-6 5.66 CXCL5, CXCL8, PPBP granulocyte chemotaxis GO:0030104 Water homeostasis 130.0E-6 4.00 FLG, FLG2, HRNR GO:0002690 Positive regulation of 120.0E-6 3.30 CXCL5, CXCL8, PPBP leukocyte chemotaxis GO:0004867 Serine-type endopeptidase 79.0E-6 3.00 SERPINB1, SERPINB12, SPINK7 inhibitor activity

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 10 of 31 Research article Immunology and Inflammation

Table 5. Top 20 significantly enriched pathways as determined by Gene Ontology (GO) enrichment analysis in dithranol-treated lesional skin at end of treatment compared to baseline from 15 patients with psoriasis. (GO was done using Cytoscape software; Bindea et al., 2009; Shannon et al., 2003 a.o. = among others).

% Associated GOID GO term P-Value Genes Associated genes found GO:0006954 Inflammatory response 17.0E-18 6.93 IL17A, IL1B, IL20, IL22, IL36A, IL36G, IL36RN, a.o. GO:0006952 Defense response 52.0E-18 4.60 CXCL8, CXCL9, DEFB103A, DEFB4A, IFNG, IL17A, IL1B, IL20, IL22, IL36A, IL36G, IL36RN, IL4R, IRAK2, IRF1, KRT16, a.o. GO:0051707 Response to other organism 56.0E-18 6.02 CCL2, CCL20, CCL22, CD24, CD80, COTL1, CXCL13, CXCL16, CXCL8, CXCL9, DDX21, DEFB103A, DEFB4A, a.o. GO:0050663 Cytokine secretion 480.0E- 13.78 IFNG, IL17A, IL1B, IL26, IL36RN, IL4R, LYN, 18 MMP12, NOS2, PAEP, PANX1, PNP, S100A12, S100A8, S100A9, a.o. GO:0001816 Cytokine production 680.0E- 6.76 IDO1, IFNG, IL17A, IL1B, IL26, IL36A, 18 IL36RN, IL4R, IRF1, LTF, LYN, MB21D1, MMP12, NOS2, a.o. GO:0012501 Programmed cell death 1.0E-15 4.05 CASP5, CASP7, CCL2, CCL21, CD24, CD274, CD38, IFNG, IL17A, IL1B, IRF1, IVL, KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, KRT74, KRT77, a.o. GO:0051240 Positive regulation of multicellular organismal 1.5E-15 4.57 CXCL17, CXCL8, FBN2, FERMT1, GBP5, GPR68, process HPSE, HRH2, IDO1, IFNG, IL17A, IL1B, IL20, IL26, IL36A, S100A8, S100A9, SERPINB3, SERPINB7, a.o. GO:0001817 Regulation of cytokine production 2.0E-15 7.04 CCL2, CCL20, CD24, CD274, CD80, CD83, CXCL17, IFNG, IL17A, IL1B, IL26, IL36A, IL36RN, IL4R, IRF1, TNFRSF9, WNT5A, a.o. GO:0002237 Response to molecule 15.0E-15 9.22 S100A8, S100A9, SELE, SOD2, TIMP4, of bacterial origin TNFRSF9, TNIP3, WNT5A, ZC3H12A, a.o. GO:0070268 Cornification 100.0E- 17.70 DSC2, DSG3, IVL, KLK13, KRT16, KRT17, 15 KRT31, KRT6A, KRT6C, KRT73, KRT74, KRT77, PI3, SPRR2A, SPRR2B, SPRR2D, a.o. GO:0043588 Skin development 190.0E- 8.17 IL20, IVL, KLK13, KRT16, KRT17, KRT31, 15 KRT6A, KRT6C, KRT73, KRT74, KRT77, LCE3A, LCE3C, LCE3E, a.o. GO:0008544 Epidermis development 220.0E- 7.63 DSC2, DSG3, EPHA2, FERMT1, FOXE1, 15 FURIN, HPSE, IL20, IVL, KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, KRT74, KRT77, a.o. GO:0009617 Response to bacterium 240.0E- 6.63 CCL2, CCL20, CD24, CD80, CXCL13, 15 CXCL16, CXCL8, CXCL9, DEFB103A, DEFB4A, S100A12, S100A8, S100A9, TREM1, a.o. GO:0001819 Positive regulation of 1.4E-12 7.89 CCL2, CCL20, CD274, CD80, CD83, cytokine production CXCL17, FERMT1, GBP5, HPSE, IDO1, IFNG, IL17A, IL1B, IL26, IL36A, a.o. GO:0050707 Regulation of cytokine secretion 2.4E-12 13.10 AIM2, CASP5, CD274, FERMT1, GBP1, IFNG, IL17A, IL1B, IL26, IL36RN, IL4R, LYN, MMP12, PAEP, PANX1, a.o. GO:0005125 Cytokine activity 4.4E-12 10.64 CCL20, CCL21, CCL22, CCL4L2, CXCL13, CXCL16, CXCL8, CXCL9, FAM3D, IFNG, IL17A, IL1B, IL20, IL22, IL26, a.o. GO:0031424 Keratinization 21.0E-12 10.48 IVL, KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, KRT74, KRT77, LCE3A, LCE3C, LCE3E, PI3, SPRR2A, SPRR2B, a.o. GO:0002790 Peptide secretion 97.0E-12 6.25 CD274, CD38, DOC2B, FAM3D, FERMT1, GBP1, GBP5, GLUL, GPR68, IFNG, IL17A, IL1B, IL26, IL36RN, IL4R, LYN, MMP12, NOS2, TREM1, WNT5A, a.o. Table 5 continued on next page

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 11 of 31 Research article Immunology and Inflammation Table 5 continued

% Associated GOID GO term P-Value Genes Associated genes found GO:0032940 Secretion by cell 140.0E- 4.04 AIM2, AMPD3, CASP5, IL36RN, IL4R, 12 LCN2, LRG1, LTF, LYN, MMP12, NOS2, NR1D1, NR4A3, OLR1, PAEP, PANX1, PLA2G3, a.o. GO:0009913 Epidermal cell differentiation 190.0E- 7.91 DSC2, DSG3, EPHA2, FURIN, IL20, IVL, 12 KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, LCE3E, PI3, SLITRK6, SPRR2A, SPRR2B, SPRR2D, SPRR2E, a.o.

mouse-tail test has evidently limitations since the disturbed cell differentiation of this model only reflects one of many aspects of psoriasis, it supports the primary effect of dithranol on keratinocytes with induced induction of orthokeratosis.

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Figure 3. Topical application of dithranol ameliorates psoriasis-like skin lesions in c-Jun/JunB knockout mice. (A) Schematic representation of experimental set-up. Red arrows indicate dithranol application in series of increasing concentrations. (Exp. 1 and 2 = experiment 1 and 2) (B) Macroscopic ear thickness on day 1 compared to day 7. Dithranol treatment led to a significant reduction in ear thickness. Controls (n = 11), dithranol group (n = 11); Paired t-test was used for statistics. (C) Representative H and E images of untreated and dithranol-treated ears. (D) Dithranol treatment led to a significant reduction in epidermis thickness. Controls (n = 10), dithranol group (n = 11); unpaired t-test was used for statistics. Data from the two experiments was pooled (D). Bars represent mean ± SD; n.s. = not significant; *p0.05; **p0.01; ***p0.001; ****p0.0001; scale bar = 100 mm. The online version of this article includes the following source data and figure supplement(s) for figure 3: Source data 1. Values displayed in scatter plots shown in Figure 3. Figure supplement 1. Strong inducing effect of dithranol in the mouse-tail test. Figure supplement 2. Gene expression analysis by RT-PCR of keratinization markers (Flg, Ivl, Krt16, Lce3e, Serpinb3a), AMPs (Lcn2, S100a8, S100a9, Camp, Defb1, Defb3) and inflammatory markers (Il1b, Il17, Il22, Cxcl1, Cxcl5) of dithranol- and vehicle-treated murine tail skin. Figure supplement 3. Dithranol causes exacerbation of psoriasis-like skin lesions in imiquimod mouse model.

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Table 6. Top 45 differentially regulated genes in dithranol-treated psoriatic skin of c-Jun/JunB knockout mice compared to that of vehicle-treated controls (p<0.05, fold change (FC) >1.5). Probe set ID Gene symbol Gene title Fold change TC0Y00000006.mm.2 Eif2s3y Eukaryotic translation initiation factor 2, 10,37 subunit 3, structural gene Y-linked TC0Y00000233.mm.2 Uty Ubiquitously transcribed tetratricopeptide 6,38 repeat gene, Y chromosome TC0Y00000235.mm.2 Ddx3y DEAD (Asp-Glu-Ala-Asp) 5,40 box polypeptide 3, Y-linked TC0900000047.mm.2 Mmp3 Matrix metallopeptidase 3 3,64 TC0Y00000079.mm.2 Ssty2 Spermiogenesis specific transcript on the Y 2 3,31 TC0100001632.mm.2 Ifi209 Interferon activated gene 209 3,23 TC0500002755.mm.2 Cxcl9 Chemokine (C-X-C motif) ligand 9 2,85 TC0300003133.mm.2 Ifi44 Interferon-induced protein 44 2,69 TC0100003591.mm.2 Ifi213 Interferon activated gene 213 2,53 TC0100001634.mm.2 Ifi208 Interferon activated gene 208 2,49 TC1900000217.mm.2 Ms4a4c Membrane-spanning 4-domains, 2,45 subfamily A, member 4C TC0300002684.mm.2 Chil3 Chitinase-like 3 2,39 TC0100003550.mm.2 Slamf7 SLAM family member 7 2,33 TC0500000922.mm.2 Cxcl13 Chemokine (C-X-C motif) ligand 13 2,26 TC1900000500.mm.2 Ifit2 Interferon-induced protein with 2,20 tetratricopeptide repeats 2 TC0500002840.mm.2 Plac8 Placenta-specific 8 2,20 TC0700001630.mm.2 Adm Adrenomedullin 2,15 TC1900000501.mm.2 Ifit3 Interferon-induced protein with 2,13 tetratricopeptide repeats 3 TC1800000610.mm.2 Iigp1 Interferon inducible GTPase 1 2,07 TC0300001446.mm.2 Gbp3 Guanylate binding protein 3 2,07 TC0300003134.mm.2 Ifi44l Interferon-induced protein 44 like 2,05 TC1900000502.mm.2 Ifit3b Interferon-induced protein with 2,02 tetratricopeptide repeats 3B TC0400003296.mm.2 Skint5 Selection and upkeep of intraepithelial T cells 5 À2,37 TC0300000482.mm.2 Aadac Arylacetamide deacetylase À2,37 TC1100000469.mm.2 Fndc9 Fibronectin type III domain containing 9 À2,39 TC0300002399.mm.2 Lce6a Late cornified envelope 6A À2,42 TC0300002409.mm.2 Lce1i Late cornified envelope 1I À2,44 TC0300002412.mm.2 Kprp Keratinocyte expressed, proline-rich À2,44 TC0300000846.mm.2 Hrnr Hornerin À2,48 TC0300002407.mm.2 Lce1g Late cornified envelope 1G À2,49 TC1600000324.mm.2 Fetub Fetuin beta À2,53 TC1300001377.mm.2 Akr1c18 Aldo-keto reductase family 1, member C18 À2,56 TC0300002406.mm.2 Lce1f Late cornified envelope 1F À2,57 TC1500001714.mm.2 Slurp1 Secreted Ly6/Plaur domain containing 1 À2,57 TC1500002344.mm.2 Gsdmc2 Gasdermin C2 À2,59 TC1000000145.mm.2 Il20ra Interleukin 20 receptor, alpha À2,66 TC0700000443.mm.2 Cyp2b19 Cytochrome P450, family 2, À2,70 subfamily b, polypeptide 19 TC0100000103.mm.2 Ly96 Lymphocyte antigen 96 À2,89 Table 6 continued on next page

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 13 of 31 Research article Immunology and Inflammation Table 6 continued

Probe set ID Gene symbol Gene title Fold change TC0700000778.mm.2 Klk5 Kallikrein related-peptidase 5 À3,15 TC0300003252.mm.2 Clca3a2 Chloride channel accessory 3A2 À3,20 TC0900002312.mm.2 Elmod1 ELMO/CED-12 domain containing 1 À3,20 TC0300002401.mm.2 Lce1a1 Late cornified envelope 1A1 À3,26 TC0700000484.mm.2 Fcgbp Fc fragment of IgG binding protein À3,83 TC0300002405.mm.2 Lce1e Late cornified envelope 1E À4,16 TC1500002274.mm.2 Krt2 Keratin 2 À5,95

In contrast to its therapeutic effect in both keratinocyte-driven psoriasis models, the c-Jun/JunB model and mouse-tail test, dithranol did aggravate psoriatic lesions in the imiquimod model that has been solely shown to be immunologically mediated and dependent on the IL-17/IL-23 axis (van der Fits et al., 2009). In the latter model, biologics such as etanercept and anti-IL-17a agents (Liu et al., 2018), topical steroids (Sun et al., 2014) and vitamin d3 analogues (Germa´n et al., 2019) but also UVB and PUVA (Shirsath et al., 2018) were shown to have a beneficial effect. In contrast, dithranol significantly enhanced overall macroscopic skin thickness, consistent with a slight increase in epidermal hyperplasia and worsened inflammation (as measured by the density of cellular infiltrate in the dermis) (Figure 3—figure supplement 3). Dithranol treatment of healthy murine skin led to similar effects upon irritation, as it increased epidermal thickness and cellular infiltrate of the skin (data not shown). However, the irritant effect of dithranol may remain without functional anti-psoriatic rele- vance in human psoriasis (as indicated by the clinical results depicted in Figure 1—figure supplement 2 and discussed below) but might be crucial in the agent’s therapeutic action in alopecia areata (Nasimi et al., 2019; Ngwanya et al., 2017). Together, these data unambiguously demonstrate that dithranol directly acts on keratinocytes, their crosstalk with neutrophils and IL-36 signaling, with AMPs being the potential link (Figure 5). This goes also in line with the observation that Langerhans cells as another type of immune cells showed a delayed response to dithranol, with no changes during treatment and an evident increase in numbers only later on (at the follow-up visit 4–6 weeks after end of treatment). These results are consistent with previous studies performed by Swinkels et al., showing that there was no significant change in T cells or Langerhans cells during twelve days of dithranol treatment (Swinkels et al., 2002a). Our findings on dithranol’s effect on members of the IL-1 family, being beneficial for its anti-psoriatic efficacy, are well in line with recent work on blockade of IL-36 pathway as a novel strategy for the treatment of pustular psoriasis (Bachelez et al., 2019) as well as plaque-type psoriasis (Benezeder and Wolf, 2019; Mahil et al., 2017). Notably, human keratinocytes express IL-1 family members (IL-36a, IL-36b, IL-36g, IL-36Ra) and their receptor IL-36R (D’Erme et al., 2015; Foster et al., 2014; Johnston et al., 2017). Furthermore, normal human keratinocytes show increased expression of IL-1 group mRNA after treatment with psoriasis-associated cytokines (TNFa, IL-1a, IL-17,IL-22) (Johnston et al., 2011). Genome-wide association studies revealed that deficiency in interleukin-36 receptor antagonist due to IL36RN mutations was associated with generalized pustular psoriasis (GPP) (Marrakchi et al., 2011; Sugiura et al., 2013). Supporting this notion, blocking IL-36 receptor was effective in reducing epidermal hyperplasia and showed comparable effects to etanercept in a psoriatic skin xenotransplantation model (Blumberg et al., 2010). Furthermore, suc- cessful treatment of psoriasis with the anti-psoriatic standard treatment etanercept is accompanied by a decrease in IL36A, IL36G and IL36RN expression (Johnston et al., 2011). A recent clinical phase one study provided proof-of-concept for the efficacy of BI 655130, a monoclonal antibody against the interleukin-36 receptor in the treatment of generalized pustular psoriasis (Bachelez et al., 2019). Intriguingly, topical short-contact dithranol therapy is efficacious not only in plaque-type psoriasis, but under certain conditions (i.e. after stabilization of disease activity with bland emollients) report- edly also in pustular psoriasis (Farber and Nall, 1993; Gerritsen, 1999). In this neutrophilic-driven condition, in which one might expect that dithranol worsens a heavy inflammatory state, it may have

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 14 of 31 Research article Immunology and Inflammation

Table 7. Top 20 significantly enriched pathways as determined by Gene Ontology (GO) enrichment analysis in dithranol-treated psoriatic skin of c-Jun/JunB knockout mice compared to that of vehicle-treated controls. (GO was done using Cytoscape software Bindea et al., 2009; Shannon et al., 2003; a.o. = among others).

% Associated Go id GO term P-Value Genes Associated genes found GO:0043588 Skin development 14,0E-18 11,11 Casp14, Cldn1, Flg2, Gjb3, Hrnr, Ivl, Krt2, Lce1a1, Lce1a2, Lce1l, Lce1m, Lor, Pou2f3, Ptgs1, Scel, Tfap2b, a.o. GO:0008544 Epidermis development 73,0E-15 9,18 Acer1, Alox8, Casp14, Cnfn, Cst6, Dnase1l2, Hrnr, Ivl, Krt2, Lce1c, Lce1d, Lce1e, Lce1f, Lce1g, Lce1h, Lce1i, Lce1j, a.o. GO:0071345 Cellular response to cytokine stimulus 1,7E-12 6,26 Ccdc3, Ccl2, Ccl5, Ccr9, Cxcl13, Cxcl9, Edn1, Il18, Il1f5, Il1f8, Il1rl1, Il20ra, Stat2, a.o. GO:0034097 Response to cytokine 7,1E-12 5,62 Cd38, Chad, Cldn1, Csf3, Edn1, Gbp2, Gbp3, Gbp4, Gbp7, Gbp8, Gbp9, Gm4951, Ifi203, Ifi204, Ifi209, Ifit1, Ifit2, Ifit3, Ifit3b, Xaf1, a.o. GO:0035456 Response to interferon-beta 12,0E-12 25,00 Gbp2, Gbp3, Gbp4, Gm4951, Ifi203, Ifi204, Ifi209, Ifit1, Ifit3, Iigp1, Irgm1, Xaf1 GO:0006952 Defense response 63,0E-12 4,11 Ccl2, Ccl5, Cd180, Cd59a, Cxcl13, Cxcl9, Cybb, Defb6, Drd1, Herc6, Hp, Il18, Il1f5, Il1f8, Il1rl1, Irgm1, Kalrn, Klk5, a. o. GO:0030855 Epithelial cell differentiation 430,0E- 5,51 Casp14, Cdkn1a, Cldn1, Cnfn, Dlx3, Dnase1l2, 12 Gsdmc2, Gstk1, Hrnr, Ivl, Klf15, Krt2, Lce1a1, Lor, Pou2f3, a.o. GO:0020005 Symbiont-containing vacuole 1,4E-9 66,67 Gbp2, Gbp3, Gbp4, Gbp7, Gbp9, Iigp1 membrane GO:0044216 Other organism cell 5,6E-9 30,77 C4b, Gbp2, Gbp3, Gbp4, Gbp7, Gbp9, Iigp1, Tap1 GO:0044406 Adhesion of symbiont to host 120,0E-9 37,50 Ace2, Gbp2, Gbp3, Gbp4, Gbp7, Gbp9 GO:0045087 Innate immune response 390,0E-9 4,35 Ccl2, Ccl5, Cd180, Cfb, Cldn1, Ifit3, Iigp1, Il1f5, Il1f8, Irgm1, Lbp, Ly96, Mx1, Parp9, Sla, Slamf6, Slamf7, Stat2, Tlr7, Trim62, a.o. GO:0034341 Response to interferon-gamma 1,0E-6 10,58 Ccl2, Ccl5, Cldn1, Edn1, Gbp2, Gbp3, Gbp4, Gbp7, Gbp8, Gbp9, Parp9 GO:0006954 Inflammatory response 5,9E-6 4,29 C3, C4b, Ccl2, Ccl5, Cd180, Cd59a, Chil3, Crip2, Ctla2a, Cxcl13, Cxcl9, Cybb, Hp, Il18, Il1f5, Il1f8, Il1rl1, Lbp, Ly96, a.o. GO:0042832 Defense response to protozoan 9,4E-6 19,35 Gbp2, Gbp3, Gbp4, Gbp7, Gbp9, Iigp1 GO:0035457 Cellular response to interferon-alpha 21,0E-6 36,36 Ifit1, Ifit2, Ifit3, Ifit3b GO:0030414 Peptidase inhibitor activity 22,0E-6 6,19 C3, C4b, Cst6, Ctla2b, Fetub, R3hdml, Serpinb12, Serpinb13, Serpinb2, Serpinb7, Spink14, Tfap2b, Wfdc12, Wfdc5 GO:0098542 Defense response to other organism 34,0E-6 4,05 Adm, Ccl5, Cxcl13, Cxcl9, Defb6, Gbp2, Gbp3, Hp, Ifit1, Ifit2, Ifit3, Ifit3b, Iigp1, Il1f5, Klk5, Lbp, Mx1, Oas1f, Plac8, Stat2, Tlr7, Wfdc12, a.o. GO:0031424 Keratinization 43,0E-6 15,00 Casp14, Cnfn, Hrnr, Ivl, Krt2, Lor GO:0044403 Symbiosis, encompassing 55,0E-6 4,11 Ace2, Acta2, Atg16l2, Ccl5, Cxcl9, Gbp2, Gbp3, mutualism through parasitism Gbp4, Gbp7, Gbp9, Ifit1, Ifit2, Ifit3, Ifit3b, Lbp, Mx1, Oas1f, Pou2f3, Rab9, Stat2, Tap1, Tlr7, Trim62

beneficial capacity by targeting the IL-36 pathway and neutrophils, both playing a most crucial role in this type of psoriasis (Bachelez, 2018; Bachelez et al., 2019; Johnston et al., 2017; Marrakchi et al., 2011; Sugiura et al., 2013). As outlined above, we observed a strong effect of dithranol on keratinocyte-neutrophil crosstalk. Importantly, early response to anti-IL-17a blockade, as one of the most effective biological treatments currently available, has been linked to inhibition of keratinocyte-neutrophil crosstalk (Reich et al., 2015). Similar to our observation, anti-IL-17a treatment with secukinumab significantly reduced epidermal hyperproliferation after 2 weeks, decreased mRNA expression of keratinocyte- derived chemotactic factors and led to a strong decrease in IL-17a positive neutrophils. Apparently,

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 15 of 31 Research article Immunology and Inflammation

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Figure 4. Venn diagram (created using InteractiVenn Heberle et al., 2015) showing comparison and overlap between differentially expressed genes (DEGs) in dithranol-treated human psoriatic skin at week 2–3 vs. day 0, DEGs in dithranol-treated human psoriatic skin at day 6 vs. day 0 and DEGs in dithranol-treated c-Jun/JunB psoriatic skin. The online version of this article includes the following figure supplement(s) for figure 4: Figure supplement 1. Verification of microarray gene expression analysis: Gene expression analysis by RT-PCR of selected genes based on microarray data in dithranol-treated c-Jun/JunB knockout mice vs. vehicle-treated controls.

disrupting the crosstalk between keratinocytes and neutrophils may be a crucial early effect in the clinical efficacy of secukinumab in psoriasis (Reich et al., 2015). In addition, decreased expression of AMPs like ß-defensin and S100 proteins was observed after only 1 week of anti-IL-17a treatment with secukinumab (Krueger et al., 2019) and after 2 weeks of ixekizumab treatment (Krueger et al., 2012), well in line with our observed early effect of dithranol on AMPs. Krueger et al. concluded that clinical efficacy of anti-IL-17a treatment is closely linked to early inhibition of keratinocyte-derived products such as chemokines and AMPs. Together this suggests that dithranol’s direct action on keratinocytes at the molecular level may disrupt IL-17 pathway dysregulation (without directly blocking IL-17 or its receptor), leading in turn to similar downstream effects as treatment with anti-IL-17 antagonists. Our study also negates the paradigm that dithranol-induced irritation is crucial for its anti-psoriatic action (Gerritsen, 1999; Kucharekova et al., 2005; Prins et al., 1998; van de Kerkhof, 1991; Wiegrebe and Mu¨ller, 1995). As depicted in Figure 1—figure supplement 2, there was no correlation between dithranol-induced perilesional as well as lesional erythema and its anti-psoriatic effect,

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 16 of 31 Research article Immunology and Inflammation

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Figure 5. Proposed model of dithranol’s mechanism of action in psoriasis. Dithranol decreases expression of keratinocyte differentiation regulators (involucrin and serpins), IL-36-related genes, keratinocyte-derived antimicrobial peptides (AMPs) (S100A7/A12 and ß-defensins) and chemotactic factors for neutrophils (CXCL5, CXCL8). This disrupts the crosstalk between keratinocytes and neutrophils and leads to diminished neutrophilic infiltration, thereby halting the inflammatory feedback loop in psoriasis. Together, this results in clearance of psoriatic lesions.

indicating that dithranol’s irritation (Prins et al., 1998; Swinkels et al., 2002c; Swinkels et al., 2002b) is a treatment side-effect unrelated to its therapeutic mechanism. However, this irritant effect of dithranol may be crucial for its action in alopecia areata, a condition, in which it was shown to be greatly effective, leading to hair regrowth in a high percentage of cases (Ngwanya et al., 2017). Similar to other topical treatment options (Nasimi et al., 2019; Yoshimasu and Furukawa, 2016), an initial irritant reaction to dithranol is followed by regrowth of hair within weeks after treatment. What our work does not answer, is how dithranol exactly acts at the molecular level. Cell culture studies have shown that dithranol targets mitochondria (McGill et al., 2005), alters cellular metabo- lism (Hollywood et al., 2015) and induces apoptosis in keratinocytes (George et al., 2013; McGill et al., 2005). Dithranol also inhibits human monocytes in vitro, inhibiting secretion of IL-6, IL- 8 and TNFa (Mrowietz et al., 1992; Mrowietz et al., 1997). Its effects on neutrophils were also shown in vitro, where dithranol leads to an increase in superoxide generation and a decrease in leukotriene production in neutrophils (Kavanagh et al., 1996; Schro¨der, 1986). Moreover, the potential receptor of dithranol remains undefined. Anti-psoriatic effects of other topical treatment options have recently been linked to modulation of the aryl hydrocarbon receptor (AhR) (Smith et al., 2017) and AhR may play a role in pathogenesis of psoriasis (Di Meglio et al., 2014). However, it appears that dithranol does not act via modulation of AhR, as we did not observe differences in the response of skin to dithranol comparing AhR knockout mice to controls (data not shown). There is need for

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 17 of 31 Research article Immunology and Inflammation further studies investigating how dithranol exactly acts on the molecular level, which receptor it potentially binds to or inhibits or whether it acts through modulation of a specific transcription factor such as NF-kB or STATs. Another question is whether the effect of dithranol is specific compared to other topical treatments such as steroids and vitamin D3 analogues. There seem to be some overlap- ping mechanisms between dithranol and vitamin D3 analogues such as calcipotriol that has been shown to act on keratinocytes to repress the expression of IL-36a/g, an effect mediated through ker- atinocytic vitamin D receptor (Germa´n et al., 2019). Moreover, similar to dithranol, calcipotriol decreased expression of AMPs such as ß-defensins in keratinocytes of psoriatic plaques (Peric et al., 2009). At the same time, calcipotriol normalized the proinflammatory cytokine milieu and decreased IL-17A, IL-17F and IL-8 transcript abundance in lesional psoriatic skin. Calcipotriol also directly targets Th17 cells (Fujiyama et al., 2016) and CD8+IL-17+ cells (Dyring-Andersen et al., 2015), whereas we found that dithranol only has delayed effects on T cells. Moreover, cathelicidin (LL37) expression was increased by calcipotriol (Peric et al., 2009), juxtaposing the results of dithranol treatment at least in the mouse-tail test of present study. That dithranol and vitamin D3 analogues may have similar basic mechanisms of action is also supported by the notion that depending on the concentration, both dithranol (Nasimi et al., 2019; Ngwanya et al., 2017) and calcipotriol (El Taieb et al., 2019; Fullerton et al., 1998; Molinelli et al., 2020) can be irritant to the skin and induce hair regrowth in alopecia areata. Compared to vitamin D3 analogues topical steroids have an even broader mechanisms of action in psoriasis linked to their anti-inflammatory, antiproliferative, vaso- constrictive (Uva et al., 2012) and immunomodulatory properties, in particular suppressing the IL- 23/IL-17 axis, with IL-23 produced by dendritic cells/macrophages and IL-17 produced by Th17 cells/ gd T cells/innate lymphoid cells (Germa´n et al., 2019). Another question that this study does not answer is, whether topical dithranol therapy has any effects on systemic psoriatic inflammation. However nowadays, treatment with dithranol is mainly administered in refractory, circumscribed psoriatic lesions in patients who do not have significant systemic inflammation how it may be otherwise the case in patients with moderate to severe forms of psoriasis. Together our work opens up several avenues for novel topical (and potentially also systemic) treatment strategies in psoriasis. Not only targeting the IL-36 pathway, but also keratinocyte differentiation regulators (e.g. involucrin), keratinocyte-produced AMPs (ß-defensins like DEFB4A, DEFB4B, DEFB103A, S100 proteins like S100A7, S100A12), and neutrophils and their chemotactic factors (CXCL5 and CXCL8) or members of the serpin family (SERPINB7 and SERPINB13), are prom- ising approaches. Such approaches may not only be helpful for chronic plaque-psoriasis, but also for pustular psoriasis, in which a vicious loop between AMPs such as cathelicidin (LL-37) and IL-36 signaling may drive psoriatic disease (Benezeder and Wolf, 2019; Furue et al., 2018; Li et al., 2014; Madonna et al., 2019; Ngo et al., 2018).

Materials and methods

Key resources table

Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-CD1a; Immunotech, Clone: O10 (undiluted) mouse monoclonal Beckman Coulter RRID:AB_10547704 Antibody anti-CD3; Novocastra, Clone: PS1 (1:100) mouse monoclonal Leica Biosystems RRID:AB_2847892 Antibody anti-CD4; Novocastra, Clone: 1F6 (1:30) mouse monoclonal Leica Biosystems RRID:AB_563559 Antibody anti-CD8; Dako, Agilent Clone: C8/144b (1:25) mouse monoclonal RRID:AB_2075537 Antibody anti-CK16; Abcam Clone: EPR13504 (1:1000) rabbit monoclonal RRID:AB_2847885 Antibody anti-FoxP3; Bio-Rad Clone: 236A/E7 (1:100) mouse monoclonal RRID:AB_2262813 Continued on next page

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Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-Ki-67; Dako, Agilent Clone: MIB-1 (1:50) mouse monoclonal RRID:AB_2631211 Antibody anti-MPO; Dako, Agilent Clone: MPO-7 (1:100) mouse monoclonal RRID:AB_578599 Strain, strain BALB/c, wild-type Charles River RRID:IMSR_CRL:028 background Laboratories Charles River Mus musculus Strain code#: 028 Strain, strain JunBf/f c-Junf/f K5-Cre-ERT PMID:16163348 Obtained from the laboratory background of Maria Sibilia (Medical Mus musculus University of Vienna) Commercial miRNeasy Mini Kit Qiagen Cat #: 217004 assay or kit Commercial iScript Reverse Bio-Rad Cat #: 1708841 assay or kit Transcription Supermix Commercial GoTag qPCR Master Mix Promega Cat #: A6001 assay or kit Commercial Human GeneChip Affymetrix, Cat #:902113 assay or kit 2.0 ST arrays ThermoFisher Scientific Commercial mouse Clariom S Assay Affymetrix, Cat #:902919 assay or kit ThermoFisher Scientific Commercial GeneChip WT ThermoFisher Scientific Cat #: 902280 assay or kit PLUS Reagent Kit Commercial GeneChip WT ThermoFisher Cat #: 900671 assay or kit Terminal Labeling Kit Scientific Commercial GeneChip Hybridization, ThermoFisher Cat #: 900720 assay or kit Wash and Stain Kit Scientific Commercial nCounter GX NanoString Custom codeset (80 target assay or kit Custom codeset Technologies genes, four reference genes Chemical Aldara (Imiquimod) MEDA Pharmaceuticals Cat #: 111981 compound, drug 5% cream Chemical Tamoxifen Sigma-Aldrich Cat #: T5648 compound, drug Chemical Dithranol (1,8-Dihydroxy- Gatt-Koller GmbH Cat #: 8069994 Dissolved in vaseline and compound, drug 9(10H)-anthracenone) Pharmaceuticals provided by the pharmacy of the Medical University of Graz, Austria Software, algorithm GraphPad GraphPad RRID:SCR_002798 Prism version 8 https://www.graphpad. com/scientific-software/prism/ Software, algorithm Interacti Venn PMID:25994840 http://www.interactivenn.net/ Software, algorithm Cytoscape PMID:19237447 RRID:SCR_003032 https://cytoscape.org/ Software, algorithm Transcriptome Analysis ThermoFisher https://www.thermofisher. Console (TAC) 4.0.2 Scientific com/at/en/home/life-science/microarray- analysis/microarray-analysis- instruments-software- services/microarray-analysis- software/affymetrix-transcriptome- analysis-console- software.html Software, algorithm Partek Genomics Partek Inc RRID:SCR_011860 Suite version 6.6 https://www.partek. com/partek-genomics-suite/ Continued on next page

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Reagent type (species) or resource Designation Source or reference Identifiers Additional information Software, algorithm R Version 3.5.1 The R Project for RRID:SCR_001905 Statistical https://www.r-project.org/ Computing Software, algorithm nSolver 2.5 Software NanoString https://www.nanostring. Technologies com/products/analysis- software/nsolver

Patients Trial protocol and patient characteristics For the clinical dithranol study, inclusion criteria were diagnosis of chronic plaque psoriasis, and age above 18 years. Exclusion criteria were intolerance of dithranol, autoimmune diseases, general poor health status, pregnancy and breast-feeding, topical treatment (steroids, vitamin D3-analogs and/or Vitamin A acid-derivates) within 2 weeks, and phototherapy within 4 weeks prior to study enrollment. None of the patients had received systemic treatment in the past prior to study enrollment. In total, 15 psoriasis patients (11 men, 4 women; median age 40.5 years, range 19.8–76.9 years) were enrolled. Mean duration of psoriasis had been 17.9 years (SD 11.5 years). Mean duration of dithranol treatment was 15.4 days (SD 3.6 days) and treatment was prematurely discontinued in two patients. Samples of normal skin from patients undergoing surgery for removal of benign skin lesions were available from 12 subjects (median age was 36.3 years, range 22.6–46.9 years) for control purposes. The samples were from lesion-adjacent, excised skin of patients who did not suffer from psoriasis, other inflammatory diseases or autoimmune diseases.

Patient treatment Dithranol ointment was prepared in the hospital pharmacy with 2% salicylic acid and white vaseline as base. It was administered to the patients daily in increasing concentrations; dosage was adjusted individually to the level of skin irritation. Concentration was usually increased every other day (starting from 0.1%, next 0.16%, 0.2%, 0.4%, 1% and finally 2%) and mean treatment duration was 15.4 days.

Marker lesions and scoring At each of the four visits (see Figure 1), i) Psoriasis Area and Severity index (PASI), ii) local psoriasis severity index (PSI) and erythema score of marker lesions and iii) perilesional erythema score were assessed. PSI was composed by rating of erythema, induration and scaling, each on a scale from 0 to 4, resulting in a maximum score of 12. Erythema score was determined by rating intensity of lesional erythema on a scale of 0–4 (0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe erythema). For perilesional erythema score, intensity of perilesional erythema was rated on a scale of 0– 3 (0 = none, 1 = mild, 2 = moderate, 3 = severe perilesional erythema). Per patient, four marker lesions of similar morphology and size and, if possible, from the same body regions or four marker areas (each 5 cm in diameter) of one or more larger psoriatic lesions were defined at study entry and then scored, treated with dithranol and later biopsy-sampled at certain timepoints.

Patient tissue sampling Biopsy samples were taken from the psoriasis patients before (day 0), during early treatment at first strong perilesional inflammation between day 4 and 9 (with most biopsies taken at day 6) at end of treatment (approximately after 2–3 weeks) and at a follow-up visit (4–6 weeks after end of therapy). Per patient, a total of five biopsy samples were taken on four study days by means of a punch cylin- der (up to 5 mm) under local anesthesia. At the first visit before starting therapy, one biopsy sample was taken additionally from adjacent non-lesional skin, with a distance of at least 5 cm from the edge of psoriatic skin. One part of each biopsy was fixed in 4% neutral-buffered paraformaldehyde and used for histology and immunohistochemistry. The other part was stored in RNAlater solution (Invitrogen, California, USA) at À80˚C until RNA extraction for further analysis.

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 20 of 31 Research article Immunology and Inflammation Histology and immunohistochemistry Analysis of HE stained sections Human and murine samples fixed with paraformaldehyde were processed routinely, cut in 4 mm sections and stained with hematoxylin and eosin (HE). Five randomly selected fields per slide were investigated for histological analysis. Thickness of epidermis was measured from basal layer to stra- tum corneum using an Olympus BX41 microscope (Olympus Life Science Solutions, Hamburg, Ger- many), cellSens software (Olympus Life Science Solutions) and 20x magnification. Semi-quantitative scoring (0 = none, 0.5 = none/low, 1 = low, 1.5 = low/moderate, 2 = moderate, 2.5 = moderate/ high, 3 = high density of infiltrate) was performed at five randomly selected locations per slide and at 20x magnification. In the mouse-tail model, degree of orthokeratosis was analyzed as described by Bosman et al., 1992 . In brief, five randomly selected scales per sample were examined and the length of the gran- ular layer (A) as well as the total length of the scale (B) were measured using cellSens software (Olympus-lifescience, Hamburg, Germany) and 20x magnification. The proportion of (A/B) x 100 depicts the percentage of orthokeratosis per scale.

Immunohistochemistry stainings and analysis Antigen retrieval was performed using either EDTA-buffer (CD1a, CD3, CD4, CK16, CD8), citrate- buffer (FoxP3, Ki-67) or trypsin (MPO). Primary antibodies used were: anti-human CD1a (mouse monoclonal, clone O10, undiluted; Immunotech, Beckman Coulter, Praque, Czech Republic), anti- human CD3 (mouse monoclonal, clone PS1, dilution 1:100; Novocastra, Leica Biosystems, Man- nheim, Germany), anti-human CD4 (mouse monoclonal, clone 1F6, dilution 1:30; Leica Biosystems), anti-human CD8 (mouse monoclonal, clone C8/144b, dilution 1:25, Dako Omnis, Agilent, Santa Clara, CA, USA), anti-human CK16 (rabbit monoclonal, clone EPR13504, dilution 1:1000, Abcam, Cambridge, UK), anti-human FoxP3 (clone 236A/E7, AbD Serotec, Bio-Rad, Hercules, CA, USA), anti-human Ki-67 (mouse monoclonal, clone MIB-1, dilution 1:50, Dako Omnis, Agilent) and anti- human MPO (mouse monoclonal, clone MPO-7, dilution 1:100, Dako Omnis, Agilent). Stainings were performed using the Dako REALTM Detection System, Peroxidase/AEC, rabbit/mouse (Dako, Agi- lent) on the Dako Autostainer Link 48 (Dako, Agilent) according to the manufacturer’s instructions. For quantification of CD1a, CD3, CD4, FoxP3, CD8, and MPO staining, all positively stained cells with visible nucleus in five randomly selected fields (separately for epidermis and dermis) per slide were counted and results were averaged to obtain mean cell counts. To quantify Ki-67 and CK16 staining, area of positive staining was divided by epidermal area as follows: one representative image per slide was taken on an Olympus BX41 microscope (Olympus Life Science Solutions, Ham- burg, Germany) at 10x magnification using cellSens software (Olympus Life Science Solutions). TIFF images were imported into ImageJ program and pixels were converted to mm. Using the polygon sections tool, the outline of epidermis was framed, and the total area was measured. In addition, total area of particles (positively stained cells) within the epidermal area was assessed.

Gene expression analyses RNA extraction Total RNA was extracted from frozen biopsies of psoriasis patients and control subjects. To facilitate homogenization, tissues were cut in 20 mm sections using a cryomicrotome and collected in pre- cooled MagNA Lyser Green Beads tubes (Roche, Basel, Switzerland) and disruption of tissue was performed on the MagNA Lyser Instrument (Roche, Basel, Switzerland). After efficient homogenization, total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. To ensure complete DNA removal, on-column DNase digestion was performed and RNA was eluted in 15–20 ml RNase-free water. Mouse tissues were handled in the same way except that sufficient homogenization of samples was obtained without using a cryomicrotome.

Microarray and pathway analysis Isolated RNA was quality checked on a Bioanalyzer BA2100 (Agilent; Foster City, CA) using the RNA 6000 Nano LabChip according to manufacturer’s instructions and samples with RNA integrity numbers (RIN) between 5 to 8 were considered for analysis using Human GeneChip 2.0 ST arrays

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 21 of 31 Research article Immunology and Inflammation (Affymetrix, ThermoFisher Scientific, Waltham, MA, USA; Cat.No.: 902113) for the human samples and mouse Clariom S Assay (Affymetrix, ThermoFisher Scientific, Waltham, MA, USA; Cat No. 902919) for the mouse samples. For first and second strand cDNA synthesis 500 ng total RNA were used in the GeneChip WT PLUS Reagent Kit (ThermoFisher) according to manufacturer’s instructions. Fragmentation and terminal labelling was performed using the GeneChipTM WT Terminal Labeling Kit (ThermoFisher) and hybridization, wash and stain of arrays was performed on a Gene- Chip Fluidics 450 station using the GeneChip Hybridization, Wash and Stain Kit (ThermoFisher) according to manufacturer’s instructions. Arrays were scanned in a GeneChip GCS300 7G Scanner and analysed with the Affymetrix Expression Console Software 1.3.1 (ThermoFisher) for the human array and Transcriptome Analysis Console (TAC) 4.0.2 for the mouse arrays. Samples were processed at the Core Facility Molecular Biology at the Centre of Medical Research at the Medical University of Graz, Austria. Pre-processing of CEL-files for the human arrays was performed with Partek Genomics Suite version 6.6 (Partek Inc, St Louis, MO, USA) using the robust multi-chip analysis (RMA) algorithm, which includes background adjustment, quantile normalisation and median polished probe summarisation. For statistical analysis, paired-sample t-tests were used and genes with p<0.05 and a fold change of at least 1.5 were considered to be significantly deregulated. All microarray data has been deposited at the public repository Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih. gov/geo/) with accession numbers GSE145126 and GSE145127. For analysis of histological responders compared to non-responders, as well as mouse arrays, pre-processing and RMA normalization was done with RStudio Version 1.2.1335 (R Version 3.5.1) with MicroArrayPipeline v1.0 Shiny app based on limma Bioconductor package for differential expression analysis and genes with p<0.05 and a fold change of at least 1.5 were considered to be significantly deregulated. Mouse and human DEGs were tested for potential overlap. Probeset IDs of early (day 6) as well as late (week 2–3) DEGs of human microarray were matched with DEGs of mouse microarray using NetAffxTM Expression Array Comparison Tool.

Nanostring nCounter analysis and microarray verification For Nanostring analysis, a nCounter GX Custom codeset (80 target genes, four reference genes, see Supplementary file 2) was designed to verify microarray results of selected DEGs. Total RNA (150 ng) with RIN values between 4.7 and 9 was used and samples were processed according to suppli- er’s instructions (NanoString Technologies, Seattle, WA USA) and scanned on a nCounter Digital Analyzer (NanoString Technologies). RCC files were used for data analysis. Raw data pre-processing and normalization was performed using nSolver 2.5 Software (NanoString Technologies) according to standard procedures (background subtraction, positive and negative controls normalization). Gene counts were then normalized to the geometric mean of the reference genes. Normalized data was uploaded to Partek Genomic Suite Software v6.6 (Partek Inc, St Louis, MO, USA) and paired t-test was used for statistical analysis. Nanostring and microarray fold change values of selected target genes were log-transformed and the two platforms were compared by Pearson correlation of each gene across samples.

Gene ontology enrichment analysis For all comparisons genes with a p-value<0.05 and FC ± 1.5 were assigned to GO enrichment analysis using Cytoscape software ver.3.5.1 (Cytoscape Consortium, NY, USA www.cytoscape.org; Bindea et al., 2009). Gene identifiers were loaded into Cytoscape software and ClueGO analysis was used to identify significantly overrepresented GO terms and associated genes. P-values (significance level <0.05) were adjusted using Bonferroni step-down corrections.

RT-qPCR Per sample 2 mg of RNA was reverse transcribed into cDNA using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA). Relative gene expression was determined using GoTag qPCR Master Mix (Promega, Mannheim, Germany) on a CFX96 Touch Real-Time PCR Detection Sys- tem (Bio-Rad). The following cycling conditions were used: Hot-start activation (95˚C, 2 min), dena- turation for 40 cycles (95˚C, 15 s) and annealing/extension (60˚C, 60 s). Melting curve analysis was done to confirm amplification specificity. For each sample, qPCRs were run in triplicates. Cycle

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 22 of 31 Research article Immunology and Inflammation thresholds (Ct) were determined and relative mRNA expression to Ubc or Ywhaz (reference genes) were calculated using the DCt method. Primer sequences are listed in Supplementary file 4.

Animals Mouse strains and housing 6–9 week-old mice were kept with food and water ad libitum in the conventional animal facility at the Centre for Medical Research, Medical University of Graz or at the Medical University of Vienna, Austria. During experiments, all mice were monitored closely to ensure sufficient health status. BALB/c mice were purchased from Charles River (Sulzfeld, Germany). c-Jun/JunB knockout mice (Zenz et al., 2005) were bred and maintained in the facilities of the Medical University of Vienna. All animal experiments were in accordance with institutional policies and federal guidelines.

Therapeutic agents used in mice For all animal experiments, dithranol in different concentrations (dissolved in vaseline) and vehicle (vaseline cream only) was provided by the pharmacy of the Medical University of Graz, Austria. Aldara (IMQ) 5% cream (MEDA Pharmaceuticals, Vienna, Austria) and tamoxifen (Sigma-Aldrich, Mis- souri, USA) were purchased.

Imiquimod model 24 hr before starting an experiment, dorsal skin of BALB/c mice was shaved carefully. To induce psoriasis-like dermatitis, imiquimod (IMQ) cream was applied daily for five consecutive days on dorsal skin (approximately 40 mg) and right ear (approximately 20 mg). A daily topical dose of 62.5 mg of IMQ cream was not exceeded; translating into 3.125 mg of the active compound. Mice received IMQ cream in the morning and dithranol treatment in the afternoon, with a time gap of 8 hr. Dithra- nol was applied topically on dorsal skin (40 mg) and right ear (20 mg) and concentrations were increased every other day (0.01% on day 1–2, 0.03% on day 3–4% and 0.1% on day 5–6). Control mice were treated similarly with Vaseline cream. Double skin fold of dorsal skin and ear thickness was measured daily in triplicates before any application using a micrometer (Mitutoyo, Kanagawa, Japan). 24 hr after the last topical treatment, mice were sacrificed and approximately 1 cm2 of dorsal skin, both ears (treated and untreated as control) were collected.

c-Jun/JunB knockout mouse model Mice carrying floxed alleles for the JunB and c-Jun locus and expressing the K5-CreERT transgene (mixed background) received consecutive i.p. injections of tamoxifen (1 mg/day) for a period of 5 days, leading to deletion of both genes in the epidermis by inducible Cre-recombinase activity (Schonthaler et al., 2009; Zenz et al., 2005). One week after the last injection, psoriasis-like lesions on the ears were treated daily with dithranol in series of increasing concentrations (0.01% on day 1– 2, 0.03% on day 3–4% and 0.1% on day 5–6 or 0.1% on day 1–2, 0.3% on day 3–4% and 1% on day 5–6) as depicted in Figure 3A. Control mice were treated similarly with vehicle cream. Ear thickness was measured daily by micrometer before any topical application and 24 hr after the last topical treatment mice were sacrificed and ears were collected.

Mouse tail test For the mouse-tail model, 40 mg dithranol 1% was applied daily for 14 days to the surface of the proximal part of tails (approx. 2 cm in length starting 1 cm from the body). 24 hr after the last application, mice were sacrificed, and treated parts of tail skin were removed from the underlying carti- lage. The mouse-tail test for psoriasis is a traditional model to quantify the effect of topical anti- psoriatics on keratinocyte differentiation by measuring degree of orthokeratosis versus parakeratosis (Bosman et al., 1992; Sebo¨k et al., 2000; Wu et al., 2015).

Statistical analyses Statistical analyses for human and murine microarrays were performed as described in the specific sections. All other statistical analyses were determined using GraphPad Prism version 8 (GraphPad software, California, USA). Normality was determined by Shapiro-Wilk test and differences between two groups were assessed by Mann Whitney test, Wilcoxon test or T-test (paired or unpaired) as

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 23 of 31 Research article Immunology and Inflammation appropriate. For multiple comparisons, One-way ANOVA with Dunnett’s test or Tukey’s test was used for parametric data and Kruskal Wallis test with Dunn’s test was used for nonparametrical data as indicated in the specific sections. Significance was set at a p-value of 0.05. Each animal experiment was performed at least twice. For correlation analysis of clinical scores, as well as comparison of microarray and nanostring ratios, Pearson or Spearman correlation was used as indicated.

Study approval A clinical study (Clinical Trials.gov no. NCT02752672) in psoriatic patients treated with dithranol was completed in cooperation with the Department of Dermatology, Klagenfurt State Hospital. Clinical trial procedures were approved by the ethics committee of the federal state of Carinthia, Austria (protocol number A23/15) and all participants gave written informed consent in accordance with the principles of the Declaration of Helsinki. All mouse experiments were approved by the Austrian Gov- ernment, Federal Ministry for Science and Research (protocol numbers BMWF-66-010/0032-11/3b/ 2018, 66.009/0200-WF/II/3b/2014) and animal experiments performed in Vienna were additionally approved by the Animal Experimental Ethics Committee of the Medical University of Vienna.

Acknowledgements The authors would like to thank all patients who made this work possible and Gerlinde Mayer andUlrike Schmidbauer, for technical support and Karin Wagner and Ingeborg Klymiuk for support in Microarray and Nanostring analyses, all Medical University of Graz; Martina Hammer, Medical University of Vienna, for support in animal experimentation; Ahmed Gehad and Rachael Clark for support in animal experimentation and scientific advice, both at Brigham and Women’s Hospital, Harvard Medical School; and Honnovara Ananthaswamy, Houston, TX, for critical reading of the manuscript. The authors would also like to thank Kathrin Eller and Herbert Strobl, who provided valuable guidance as members of the thesis committee of TB. This work was supported by the Austrian Science Fund FWF (W1241) and the Medical University of Graz through the Ph.D. Program Molecular Fundamentals of Inflammation (DK-MOLIN) to PW; by grants from the Austrian Science Fund (FWF, W1212), the WWTF-project LS16-025, the European Research Council (ERC) Advanced grant (ERC-2015-AdG TNT-Tumors 694883) and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 766214 (Meta-Can) to MS. TB was supported by the Austrian Marshall Plan Foundation and the Austrian Society for Derma- tology and Venereology (Klaus Wolff fellowship). CP was supported by the Elisabeth Hirschheiter research grant. PW was supported by a Visiting Scholar Award of the Human Skin Disease Resource Center (HSDRC) Grant Program (NIH/NIAMS P30AR069625), Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School.

Additional information

Funding Funder Grant reference number Author Austrian Science Fund W1241 Peter Wolf Medical University of Graz PhD Program Molecular Peter Wolf Fundamentals of Inflammation (DK-MOLIN) Austrian Science Fund W1212 Maria Sibilia Vienna Science and Technol- LS16-025 Maria Sibilia ogy Fund H2020 European Research ERC-2015-AdG TNT- Maria Sibilia Council Tumors 694883 Horizon 2020 Framework Pro- 766214 Maria Sibilia gramme Marshallplan-Jubila¨ umsstif- Theresa Benezeder tung

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 24 of 31 Research article Immunology and Inflammation

Austrian Society for Dermatol- Theresa Benezeder ogy and Venereology Elisabeth Hirschheiter research Clemens Painsi grant National Institute of Arthritis P30AR069625 Peter Wolf and Musculoskeletal and Skin Diseases

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Author contributions Theresa Benezeder, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration; Clemens Painsi, Concep- tualization, Data curation, Investigation, Project administration; VijayKumar Patra, Visualization, Methodology; Saptaswa Dey, Methodology; Martin Holcmann, Resources, Methodology; Bernhard Lange-Asschenfeldt, Resources, Project administration; Maria Sibilia, Resources, Writing - review and editing; Peter Wolf, Conceptualization, Supervision, Funding acquisition, Writing - review and editing

Author ORCIDs Theresa Benezeder https://orcid.org/0000-0001-6218-2792 VijayKumar Patra https://orcid.org/0000-0002-7161-7767 Saptaswa Dey https://orcid.org/0000-0001-7532-7858 Peter Wolf https://orcid.org/0000-0001-7777-9444

Ethics Clinical trial registration Clinical Trials.gov no. NCT02752672. Human subjects: A clinical study in psoriatic patients treated with dithranol was completed in cooperation with the Department of Dermatology, Klagenfurt State Hospital. Clinical trial procedures were approved by the ethics committee of the federal state of Carinthia, Austria (protocol number A23/15) and all participants gave written informed consent in accordance with the principles of the Declaration of Helsinki. Animal experimentation: All mouse experiments were approved by the Austrian Government, Fed- eral Ministry for Science and Research (protocol numbers BMWF-66-010/0032-11/3b/2018, 66.009/ 0200-WF/II/3b/2014) and animal experiments performed in Vienna were additionally approved by the Animal Experimental Ethics Committee of the Medical University of Vienna.

Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.56991.sa1 Author response https://doi.org/10.7554/eLife.56991.sa2

Additional files Supplementary files . Supplementary file 1. Top 45 upregulated genes in lesional psoriatic skin compared to non-lesional skin at baseline from 15 patients with psoriasis (p<0.05, fold change >1.5). . Supplementary file 2. Verification of microarray results of dithranol-treated skin samples of psoriasis patients with Nanostring analysis (nCounter GX Custom codeset) of 80 target genes and four reference genes. FC, fold change . Supplementary file 3. Top 20 significantly enriched pathways as determined by Gene Ontology (GO) enrichment analysis in histological responders compared to non-responders in the psoriasis patient cohort (GO was done using Cytoscape software; [Bindea et al., 2009; Shannon et al., 2003] a.o. = among others).

Benezeder et al. eLife 2020;9:e56991. DOI: https://doi.org/10.7554/eLife.56991 25 of 31 Research article Immunology and Inflammation . Supplementary file 4. qPCR Primer sequences and corresponding annealing temperatures. . Transparent reporting form

Data availability All microarray data has been deposited at the public repository Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE145126 and GSE145127.

The following datasets were generated:

Database and Author(s) Year Dataset title Dataset URL Identifier Benezeder T, Painsi 2020 Microarray analysis of c-Jun/JunB https://www.ncbi.nlm. NCBI Gene C, Patra V, Dey S, knockout mice treated with nih.gov/geo/query/acc. Expression Omnibus, Holcmann M, dithranol cgi?acc=GSE145126 GSE145126 Lange-Asschenfeldt B, Sibilia M, Wolf P Benezeder T, Painsi 2020 Microarray analysis of dithranol- https://www.ncbi.nlm. NCBI Gene C, Patra V, Dey S, treated psoriasis nih.gov/geo/query/acc. Expression Omnibus, Holcmann M, cgi?acc=GSE145127 GSE145127 Lange-Asschenfeldt B, Sibilia M, Wolf P

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